Macrolide antibiotics effective on, for example, Gram-positive bacteria, Mycoplasma and Chlamydia, are regarded as clinically important antimicrobial drugs since they can be orally administered and have low toxicity. In order to improve their clinical availability, research groups in various countries have been positively conducting studies on derivatives of 16-membered macrolide antibiotics. Several derivatives of 16-membered macrolides have been synthesized by acylating hydroxyl groups at specific positions for improving their antimicrobial activities in vitro and/or pharmacokinetics. Particularly, miokamycin [Journal of Antibiotics, 29(5), 536 (1976) and Japanese Journal of Antibiotics, 35(6), 1462 (1982)] has been frequently used clinically, mainly in the field of pediatrics, as a semisynthetic 16-membered macrolide antibiotic, because of its excellent pharmacokinetics and little bitterness in comparison with natural compounds. Recently, studies on the development of the derivatives have been directed mostly toward introduction of substituted amino groups into a lactone ring a 16-membered macrolide [Journal of Antibiotics, 44(4), 448 (1991)], deoxylation of specific hydroxyl groups [Journal of antibiotics, 45(1), 144 (1992)] or alkylation of hydroxyl groups which is significant in terms of pharmacokinetics. The 16-membered macrolide antibiotics are advantageous in that: (1) resistance to these antibiotics is scarcely induced; (2) they exert less interaction with other drugs; (3) they show less affect on the intestinal tract; and (4) they give little irritation at the oral administration. In view of these characteristics, the present inventors have selected 16-membered macrolide antibiotics as a research subject and have conducted studies on the screening of a compound which shows excellent effects on various Gram-positive bacteria, by means of synthetic chemical and biochemical approaches.
Firstly, the present inventors have found a synthetic process for introducing a methyl group to a tertiary hydroxyl group at the 3"-position of a 16-membered macrolide as a methodology for the improvement of its antimicrobial activity in vitro based on the chemical structure of L-chladinose which is a constituent sugar of erythromycin. Tatsuta et al. have reported the synthesis of another 16-membered macrolide having a methylated hydroxyl group at the 3"-position which has been prepared by a glycosylation method differing from the process provided by the present inventors [Chemistry Letters, 769 (1977)]. According to this report, 3"-O-methylcarbomycin B shows higher antimicrobial activity than carbomycin B against certain bacteria, for example, a species of acid-fast bacteria. The derivatives described in these reports, in which a hydroxyl group at the 3"-position is methylated, have a carbonyl group, an sp.sup.2 carbon, at the 9-position. No information is available to date concerning a 16-membered macrolide derivative having an sp.sup.3 carbon at the 9-position and a methylated tertiary hydroxyl group at the 3"-position.
Secondly, with regard to a microbial conversion in which a carbonyl group at the 9-position of 16-membered macrolide is reduced to a hydroxyl group having a natural configuration, its application example and efficacy obtained thereby have already reported by the present inventors (EP-A-0 595 303). In order to clarify the correlation among structures of 16-membered macrolide compounds, to study biosynthesis of these compounds and to analyze the structures thereof, there have been known methods for reducing a carbonyl group at the 9-position of a 16-membered macrolide compound into a hydroxyl group through a synthetic chemical approach [Journal of Organic Chemistry, 39(16), 2474 (1974); Journal of Antibiotics, 34(12), 1577 (1981); and ibid., 39(12), 1784 (1986)] and through a biochemical approach [JP-A-50-126880; Journal of Antibiotics, 32(7), 777 (1979); JP-A-54-8793; and Journal of Antibiotics, 33(8), 911 (1980)] (The term "JP-A" as used herein means an "unexamined published Japanese patent application").
Thirdly, with regard to a microbial conversion for specifically cleaving an acyl group binding to a hydroxyl group at the 3-position of a lactone ring of a 16-membered macrolide to form a free hydroxyl group, the present inventors have reported its details including studies on substrate specificity of the conversion reaction [EP-A-526,906 (1993), U.S. Pat. No. 5,219,736 (1993) and JP-A-6-16691]. On the other hand, similar biochemical reactions effected by Bacillus subtilis, or an enzyme produced thereby, have been already reported [Journal of Fermentation Technology, 57(6), 519 (1979) and JP-A-54-28892].
Fourthly, an investigation has been made on derivatives of Medemycin (midecamycin A.sub.1) [Journal of Antibiotics, 24(7), 452 (1971)], a naturally occurring 16-membered macrolide antibiotic, in order to improve its in vivo effect. As a result, several excellent medemycin derivatives have been obtained, including a 16-membered macrolide derivative in which a hydroxyl group at the 3"-position in the neutral sugar moiety is converted into a methylthiomethyl (--CH.sub.2 SCH.sub.3) group, which is used as a key intermediate of the present invention. A synthetic method of the derivative is reported in Journal of Antibiotics, 33(1), 61 (1980). In the field of carbohydrate chemistry, it has been known since 1960 that a methylthiomethyl group introduced into a secondary hydroxyl group is reduced to a methoxy group [Carbohydrate Research, 7, 474 (1968)].
Fifthly, the present inventors have already provided a synthetic method in which a 16-membered macrolide antibiotic having a free hydroxyl group at the 3-position of a lactone ring is used as a starting material and an alkyl group is introduced by turns into the 4"-position and 3"-position hydroxyl groups in the neutral sugar moiety (EP-A-0 595 303).
Sixthly, as to protection of a hydroxyl group at the 3-position of a lactone ring of a 16-membered macrolide derivative, there is a report on protection of a hydroxyl group at the 3-position of a lactone ring of spiramycins and tylosins with an acetal base substituent group having an asymmetric carbon atom. However, except for spiramycins, there are no synthetic intermediate compound having a platenolide skeleton (a lactone ring of leucomycin), in which a hydroxyl group at the 3-position of a lactone ring is substituted with an acetal group having an asymmetric carbon atom.
It has been reported that 16-membered macrolide antimicrobial agents cause little side effects but their antimicrobial activities in vitro are generally weak in comparison with those of 14-membered counterparts [Antimicrobial Agents and Chemotherapy, 32(11), 1710 (1988)]. In consequence, great concern has been directed in the first place toward the development of an excellent derivative of a 16-membered macrolide antibiotic, whose antimicrobial activities against the genus Streptococcus as one of the main causative bacteria of clinically important upper respiratory infection and Enterococcus feacalis as one of the causative bacteria of multiple infection with MRSA (The 461th Special Member Meeting of Japan Antibiotics Research Association, Feb. 22, 1991) are improved to the level of 14-membered new macrolides, while keeping the 16-membered macrolide's superior features to cause little side effects and drug interaction.
On the other hand, cost and time required for the production of such derivatives are not always satisfactory, because it is necessary to perform a chemical reaction consisting of a plural number of steps involving regio- and stereo-selective glycosylation for introducing a neutral sugar and two continuous steps of microbial conversion in the practice of the preparation of these derivatives. Furthermore, an activator, which is dangerous and to be handled with care, should be stoichiometrically used in the aforementioned glycosylation reaction, and there are some problems in scaling up thereof such as many days required for the production. Accordingly, it has been secondly required to establish a process for the production of a 16-membered macrolide derivative in which the 3"-position hydroxyl group in the neutral sugar moiety of the present invention is methylated, by pure chemical synthesis through less steps without using any glycosylation reaction.